Microscopic device and microscopic method for the three-dimensional localization of point-like objects

Abstract

A microscopic device provides three-dimensional localization of point-like objects and includes two imaging optics, each configured to image a same point-like object located in an object space into two separate image spaces as a focused light distribution. Two detector units are respectively associated with the imaging optics and configured to capture an analyzable light spot in detection points of a detection surface disposed in the respective image space. Each imaging optics includes an optical device that orients the focused light distributions obliquely to a detection axis such that, taking into account the detection point correspondence, the two light spots shift in opposite directions based on a z-position of the point-like object. An evaluation unit brings the detection points of the two detection surfaces into mutual pairwise correspondence and analyzes the two light spots so as to ascertain a lateral x-y position and an axial z-position of the point-like object.

What is claimed is: 
     
       1. A microscopic device for three-dimensional localization of point-like objects, the microscopic device comprising:
 two imaging optics, each configured to image a same point-like object in a single object plane located in an object space into a respective separate image space in a form of a focused light distribution; 
 two detector units, each associated with a respective one of the imaging optics and configured to capture an analyzable light spot in a detection point of a detection surface disposed in the respective image space that represents a planar cross section through the respective focused light distribution, each of the imaging optics including an optical device configured to orient the respective focused light distribution obliquely to a detection axis that is provided in the respective imaging optics and that is disposed perpendicularly to the detection surface of the respective detector unit, the obliquities of the two focused light distributions being opposite to each other in such a way that, taking into account the detection point correspondence, the two light spots shift in opposite directions in response to a change in the z-position of the point-like object in the respective detection surface; and 
 an evaluation unit configured to bring the detection points of the two detection surfaces into pairwise correspondence and to analyze the two light spots in view of the image point correspondences so as to ascertain a lateral x-y position of the point-like object within the object plane residing in the object space and an axial z-position of the point-like object in a direction of an optical axis disposed perpendicularly to the object plane, the evaluation unit ascertaining the axial z-position of the point-like object based on the relative position of the two light spots. 
 
     
     
       2. The microscopic device as recited in  claim 1 , wherein the evaluation unit is configured to acquire a centroid position of each respective light spot and determine the lateral x-y position, as well as the axial z-position on the basis of the acquired centroid positions of the two light spots. 
     
     
       3. The microscopic device as recited in  claim 2 , wherein the evaluation unit is configured to ascertain the lateral x-y position of the point-like object based on of a mean of the centroid positions of the two light spots. 
     
     
       4. The microscopic device as recited in  claim 2 , wherein the evaluation unit is configured to ascertain the axial z-position of the point-like object based on of a distance between the centroid positions of the two light spots. 
     
     
       5. The microscopic device as recited in  claim 1 , wherein the detection surfaces of each of the imaging optics is optically conjugate to the object plane in the object space. 
     
     
       6. The microscopic device as recited in  claim 1 , further comprising:
 a recording lens that is shared by both imaging optics and that is configured to convert light emerging from the point-like object into a bundle of rays, and 
 a beam splitter configured to split the bundle of rays generated by the recording lens into two partial bundles of rays which each produce one of the two light spots on the respective detection surface. 
 
     
     
       7. The microscopic device as recited in  claim 6 , wherein the rays of the bundle are parallel. 
     
     
       8. The microscopic device as recited in  claim 6 , wherein each of the imaging optics includes a tube lens configured to focus the respective partial bundle of rays onto the respective detection surface. 
     
     
       9. The microscopic device as recited in  claim 1 , wherein the optical device for obliquely angling the respective focused light distribution provides an off-center illumination of an optical element provided in the respective imaging optics. 
     
     
       10. The microscopic device as recited in  claim 9 , wherein the off-center illumination is an off-center under-illumination. 
     
     
       11. The microscopic device as recited in  claim 1 , wherein the optical device is a lens having an aspherical lens surface. 
     
     
       12. The microscopic device as recited in  claim 1 , wherein the optical device is a spatial light modulator. 
     
     
       13. The microscopic device as recited in  claim 1 , wherein the two detector units are jointly configured on one module. 
     
     
       14. A microscopic method for three-dimensional localization of point-like objects, the method comprising:
 imaging a same point-like object in a single object plane located in an object space in a form of focused light distribution into two separate image spaces; 
 capturing, for each of the image spaces, an analyzable light spot in a respective detection point of a respective detection surface disposed in a respective one of the image spaces that represents a planar cross section through a respective one of the focused light distributions, each of the respective focused light distributions being oriented obliquely to a detection axis that is disposed perpendicularly to the respective detection surface; 
 bringing the detection points of the respective two detection surfaces into pairwise correspondence, the obliquities of the two focused light distributions being opposite to each other in such a way that, taking into account the detection point correspondence, the two light spots shift in opposite directions in response to a change in the z-position of the point-like object in the respective detection surfaces; and 
 analyzing the two light spots, taking into account the detection point correspondence, so as to ascertain a lateral x-y position of the point-like object within the object plane residing in the object space and an axial z-position of the point-like object in a direction of an optical axis disposed perpendicularly to the object plane, the axial z-position of the point-like object being ascertained based on a relative position of the two light spots. 
 
     
     
       15. The microscopic method as recited in  claim 14 , wherein a plurality of point-like objects are simultaneously imaged onto each of the respective detection surfaces for three-dimensional localization, the point-like objects being spaced apart in the object space by lateral distances that are large enough to allow the light spots imaging the point-like objects in the respective detection surface to be captured spatially separately from one another. 
     
     
       16. The microscopic method as recited in  claim 15 , wherein the point-like objects are objects that are switchable between a bright and a dark state. 
     
     
       17. The microscopic method as recited in  claim 16 , wherein the objects are fluorescing molecules.